PROJECT SUMMARY Epithelial to mesenchymal transition (EMT) is a developmental program in which non-motile epithelial cells with tight cell-cell adherence convert into individual, motile mesenchymal cells. Importantly, EMT is reversible through a mesenchymal to epithelial transition (MET) converting mesenchymal cells back to an epithelial state. The EMT program is essential for normal processes during development and for tissue regeneration. EMT is reactivated in human diseases including tissue fibrosis in the kidney, liver, and lung and in cancer metastasis, making EMT a key target for drug therapy. Targeted mutation of the active site lysine of the kinase MAP3K4 in the mouse (KI4) results in severe developmental defects due to disrupted EMT. Epithelial trophoblast stem (TS) cells from kinase-inactive MAP3K4 (KI4) mice (TSKI4 cells) are uniquely paused in the intermediate stages of EMT, expressing both epithelial and mesenchymal characteristics while maintaining stemness. Induction of EMT in TSKI4 cells is due in part to the loss of MAP3K4/CBP mediated acetylation of histone H2B on the promoters of genes controlling the epithelial phenotype. We have recently discovered that loss of MAP3K4 activity increases the expression and activity of another chromatin remodeler, HDAC6. The goal of this project is to define the molecular network regulated by MAP3K4 controlling EMT. This network includes signaling pathways leading to the epigenetic regulation of genes important for EMT. Our approach is based on both our published work and new findings showing that MAP3K4 coordinates EMT by activating CBP and inhibiting HDAC6 on the promoters of genes important for EMT. We predict that genes in this MAP3K4 controlled network are critical to EMT-related pathologies. Our rationale is based on our findings of overlapping gene expression signatures between our TSKI4 stem cells and claudin low breast cancer cells that both display characteristics of stemness and EMT. Cancers with these characteristics frequently are both metastatic and display resistance to therapy. Protein networks that regulate the EMT transition represent potential targets for therapy with the rationale that reversal of EMT would restore sensitivity towards therapy. Our preliminary work using this innovative system has successfully identified new genes in breast cancer that control EMT/MET. Aim1 uses RNA-seq and ChIP-seq to identify a MAP3K4/CBP/HDAC6/H2BK5Ac dependent network that controls EMT. Aim 2 is a mechanistic study of the role of MAP3K4 and HDAC6 in EMT and the impact on the KI4 cellular and organismal phenotypes. The third aim targets GALNT3, a MAP3K4/CBP/HDAC6/H2BK5Ac co- regulated gene, defining the mechanisms by which GALNT3 controls EMT. Together, the three aims of this proposal will define the signaling mechanisms by which MAP3K4 coordinates the cellular phenotype through the co-regulation of the chromatin modifiers CBP and HDAC6. Further, this proposal will identify and characterize novel genes regulating epithelial/mesenchymal states, leading to the discovery of new biomarkers and drug targets for the identification and treatment of EMT related pathologies like fibrosis and metastasis.